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Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45–55 Skipping Accompanied by Rescue of Dystrophin Expression

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Exon Skipping and Inclusion Therapies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1828))

Abstract

Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach for the treatment of various genetic diseases and a therapy which has gained significant traction in recent years following FDA approval of new antisense-based drugs. Exon skipping for Duchenne muscular dystrophy (DMD) works by modulating dystrophin pre-mRNA splicing, preventing incorporation of frame-disrupting exons into the final mRNA product while maintaining the open reading frame, to produce a shortened-yet-functional protein as seen in milder Becker muscular dystrophy (BMD) patients. Exons 45–55 skipping in dystrophin is potentially applicable to approximately 47% of DMD patients because many mutations occur within this “mutation hotspot.” In addition, patients naturally harboring a dystrophin exons 45–55 in-frame deletion mutation have an asymptomatic or exceptionally mild phenotype compared to shorter in-frame deletion mutations in this region. As such, exons 45–55 skipping could transform the DMD phenotype into an asymptomatic or very mild BMD phenotype and rescue nearly a half of DMD patients. In addition, this strategy is potentially applicable to some BMD patients as well, who have in-frame deletion mutations in this region. As the degree of exon skipping correlates with therapeutic outcomes, reliable measurements of exon skipping efficiencies are essential to the development of novel antisense-mediated exon skipping therapeutics. In the case of DMD, researchers have often relied upon human muscle fibers obtained from muscle biopsies for testing; however, this method is highly invasive and patient myofibers can display limited proliferative ability. To overcome these challenges, researchers can generate myofibers from patient fibroblast cells by transducing the cells with a viral vector containing MyoD, a myogenic regulatory factor. Here, we describe a methodology for assessing dystrophin exons 45–55 multiple skipping efficiency using antisense oligonucleotides in human muscle cells derived from DMD patient fibroblast cells.

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Acknowledgments

This work was supported by the University of Alberta Faculty of Medicine and Dentistry, Parent Project Muscular Dystrophy USA, Canadian Institutes of Health Research (grants FRN134134 and 132574), Friends of Garrett Cumming Research Funds, HM Toupin Neurological Science Research Funds, Muscular Dystrophy Canada, Canada Foundation for Innovation (grant 30819), Alberta Enterprise and Advanced Education, Women and Children’s Health Research Institute, Association Française contre les Myopathies, Alberta Innovates—Health Solutions, NIH (grants 5U54HD053177, K26OD011171, and P50AR060836-01), US Department of Defense (grants W81XWH-05-1-0616 and W81XWH-11-1-0782), and NIH NIAMS (training grant 5T32AR056993).

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Authors and Affiliations

  1. Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada

    Joshua J. A. Lee & Toshifumi Yokota

  2. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan

    Takashi Saito & Shin’ichi Takeda

  3. Northern Ireland Centre for Stratified Medicine, Altnagelvin Hospital Campus, Ulster University, Londonderry, UK

    William Duddy

  4. The Friends of Garrett Cumming Research and Muscular Dystrophy Canada HM Toupin Neurological Science Research Chair, Edmonton, AB, Canada

    Toshifumi Yokota

Authors
  1. Joshua J. A. Lee
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  2. Takashi Saito
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  3. William Duddy
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  4. Shin’ichi Takeda
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  5. Toshifumi Yokota
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Correspondence to Toshifumi Yokota .

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Editors and Affiliations

  1. Medical Genetics, University of Alberta, Edmonton, AB, Canada

    Toshifumi Yokota

  2. Medical Genetics, University of Alberta, Edmonton, AB, Canada

    Rika Maruyama

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Lee, J.J.A., Saito, T., Duddy, W., Takeda, S., Yokota, T. (2018). Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45–55 Skipping Accompanied by Rescue of Dystrophin Expression. In: Yokota, T., Maruyama, R. (eds) Exon Skipping and Inclusion Therapies. Methods in Molecular Biology, vol 1828. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8651-4_8

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  • DOI: https://doi.org/10.1007/978-1-4939-8651-4_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8650-7

  • Online ISBN: 978-1-4939-8651-4

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